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Biological Procedures Online

, Volume 1, Issue 1, pp 70–80 | Cite as

Expression of a prokaryotic P-type ATPase in E. coli plasma membranes and purification by Ni2+-affinity chromatography

  • Markus Geisler
Open Access
Article

Abstract

In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20–25% of the membrane protein. The purification of the Synechocystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 μM) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

Keywords

ATPase Activity Plasma Membrane Vesicle Biological Procedure Calcium Pump Total Cell Extract 
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Copyright information

© Springer 1998

Authors and Affiliations

  • Markus Geisler
    • 1
  1. 1.Institut für Biochemie der PflanzenHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany

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